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Direct-acting antiviral (DAA) drugs have been shown to effectively reduce viral load and cure a high proportion of hepatitis C virus (HCV) infections. However, costs associated with the course of therapy and any possible adverse effects should also be considered. It is important to acknowledge, moreover, that certain groups may not be eligible for treatment. Given that there is currently no approved vaccine for HCV infection, the need for an effective, safe, and accessible treatment remains a crucial priority. The aim of this study is to develop an antisense oligonucleotide (ASO)-based therapeutic drug that can inhibit HCV capsid. After analyzing 817 HCV capsid protein mRNA sequences using the NCBI Virus Data Portal, a conserved region of 7 nucleotides (nt) was identified in all genotypes (1–7). However, because of its high GC% content, this region is not a suitable target for ASO. Conversely, the other highly conserved region, which is only 8 nt long, was preserved in 801 datasets after removing missing and differing sequence data. The candidate ASO was then investigated using computer simulations to assess its potential. Thus, it is possible that the ASO sequence consisting of 8 nt could be a viable therapeutic target for the inhibition of HCV capsid. Furthermore, the 7 nt sequence, which is conserved in all datasets, may be targeted using alternative strategies in lieu of ASO-based targeting. [ABSTRACT FROM AUTHOR]

Background/Objectives: Hepatitis E virus (HEV) is an RNA virus recognized to be spread mainly by fecal-contaminated water. Its infection is known to be a serious threat to public health globally, mostly in developing countries, in which Africa is one of the regions sternly affected. An African-based vaccine is necessary to actively prevent HEV infection. Methods: This study developed an in silico epitope-based subunit vaccine, incorporating CTL, HTL, and BL epitopes with suitable linkers and adjuvants. Results: The in silico-designed vaccine construct proved immunogenic, non-allergenic, and non-toxic and displayed appropriate physicochemical properties with high solubility. The 3D structure was modeled and subjected to protein docking with Toll-like receptors 2, 3, 4, 6, 8, and 9, which showed a stable binding efficacy, and the dynamics simulation indicated steady interaction. Furthermore, the immune simulation predicted that the designed vaccine would instigate immune responses when administered to humans. Lastly, using a codon adaptation for the E. coli K12 bacterium produced optimum GC content and a high CAI value, which was followed by in silico integration into a pET28 b (+) cloning vector. Conclusions: Generally, these results propose that the design of an epitope-based subunit vaccine can function as an outstanding preventive vaccine candidate against HEV, although validation techniques via in vitro and in vivo approaches are required to justify this statement. [ABSTRACT FROM AUTHOR]

The zoonotic transmission of hepatitis E virus (HEV) genotypes 3 (HEV-3) and 4 (HEV-4), and rabbit HEV (HEV-3ra) has been documented. Vaccination against HEV infection depends on the capsid (open reading frame 2, ORF2) protein, which is highly immunogenic and elicits effective virus-neutralizing antibodies. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles (VLPs). However, research on the production of ORF2 proteins from these HEV genotypes in E. coli to form VLPs has been modest. In this study, we constructed 21 recombinant plasmids expressing various N-terminally and C-terminally truncated HEV ORF2 proteins for HEV-3, HEV-3ra, and HEV-4 in E. coli. We successfully obtained nine HEV-3, two HEV-3ra, and ten HEV-4 ORF2 proteins, which were primarily localized in inclusion bodies. These proteins were solubilized in 4 M urea, filtered, and subjected to gel filtration. Results revealed that six HEV-3, one HEV-3ra, and two HEV-4 truncated proteins could assemble into VLPs. The purified VLPs displayed molecular weights ranging from 27.1 to 63.4 kDa and demonstrated high purity (74.7–95.3%), as assessed by bioanalyzer, with yields of 13.9–89.6 mg per 100 mL of TB medium. Immunoelectron microscopy confirmed the origin of these VLPs from HEV ORF2. Antigenicity testing indicated that these VLPs possess characteristic HEV antigenicity. Evaluation of immunogenicity in Balb/cAJcl mice revealed robust anti-HEV IgG responses, highlighting the potential of these VLPs as immunogens. These findings suggest that the generated HEV VLPs of different genotypes could serve as valuable tools for HEV research and vaccine development. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV), a mosquito‐borne Flavivirus of international concern, causes congenital microcephaly in newborns and Guillain–Barré syndrome in adults. ZIKV capsid (C) protein, one of three key structural proteins, is essential for viral assembly and encapsidation. In dengue virus, a closely related flavivirus, the homologous C protein interacts with host lipid systems, namely intracellular lipid droplets, for successful viral replication. Here, we investigate ZIKV C interaction with host lipid systems, showing that it binds host lipid droplets but, contrary to expected, in an unspecific manner. Contrasting with other flaviviruses, ZIKV C also does not bind very‐low density‐lipoproteins. Comparing with other Flavivirus, capsid proteins show that ZIKV C structure is particularly thermostable and seems to be locked into an auto‐inhibitory conformation due to a disordered N‐terminal, hence blocking specific interactions and supporting the experimental differences observed. Such distinct structural features must be considered when targeting capsid proteins in drug development. [ABSTRACT FROM AUTHOR]

Background/Objectives: Hepatitis E virus (HEV) is an RNA virus recognized to be spread mainly by fecal-contaminated water. Its infection is known to be a serious threat to public health globally, mostly in developing countries, in which Africa is one of the regions sternly affected. An African-based vaccine is necessary to actively prevent HEV infection. Methods: This study developed an in silico epitope-based subunit vaccine, incorporating CTL, HTL, and BL epitopes with suitable linkers and adjuvants. Results: The in silico-designed vaccine construct proved immunogenic, non-allergenic, and non-toxic and displayed appropriate physicochemical properties with high solubility. The 3D structure was modeled and subjected to protein docking with Toll-like receptors 2, 3, 4, 6, 8, and 9, which showed a stable binding efficacy, and the dynamics simulation indicated steady interaction. Furthermore, the immune simulation predicted that the designed vaccine would instigate immune responses when administered to humans. Lastly, using a codon adaptation for the E. coli K12 bacterium produced optimum GC content and a high CAI value, which was followed by in silico integration into a pET28 b (+) cloning vector. Conclusions: Generally, these results propose that the design of an epitope-based subunit vaccine can function as an outstanding preventive vaccine candidate against HEV, although validation techniques via in vitro and in vivo approaches are required to justify this statement.

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are members of a group of genetically highly homologous lentiviruses collectively referred to as small ruminant lentiviruses (SRLVs). SRLVs can infect sheep, goats and other small ruminants, causing multisystemic disease with progressive and persistent inflammatory changes, severely reducing animal productivity and impeding animal trade. The capsid protein of SRLVs, p28, is highly conserved among strains and is a commonly used marker for the detection of SRLVs. In this study, two monoclonal antibodies (mAbs), designated G8F7 and A10C12, against p28 were generated using a recombinant p28 protein expressed in Escherichia coli as an immunogen. Functional analysis showed that these two monoclonal antibodies could be used in iELISA, immunofluorescence assays (IFA) and western blot assays to detect p28 or Gag precursor proteins of SRLVs. Two linear epitopes, 61GNRAQKELIQGKLNEEA77 (E61-77) and 187CQKQMDRVLGTRVQQATVEEKMQACR212 (E187-212), which are recognized by G8F7 and A10C12, respectively, were identified through truncation of the GST-fused p28. Amino acid sequence alignment showed that the epitope E61-77 is conserved among SRLVs, with a dominant mutation site (K72R) that does not disrupt recognition by G8F7. E187-212 was found to exhibit variability among SRLVs, but the majority of mutant epitopes are recognized by A10C12, with the exception of a mutant epitope from an isolate with undefined subtypes from Ovis aries, which was not recognized. These findings may facilitate future study of SRLVs and promote the development of methods for the detection of these viruses. [ABSTRACT FROM AUTHOR]

The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein. [ABSTRACT FROM AUTHOR]

Porcine Circovirus 4 (PCV4) has been detected in pigs from China only in 2019. PCV4 has been found to be associated with several forms of clinical syndromes in pigs. Like other Porcine Circoviruses, the only structural protein of PCV4 is the capsid protein. Therefore, it could be a key vaccine candidate or diagnostic marker. The present study aims to characterize the capsid protein of PCV4 by using several bioinformatics tools. The in-silico analysis revealed that the molecular weight of PCV4 capsid protein is about 27.3 kDa which is made up of 228 amino acids. The capsid protein of PCV4 contains a nuclear localization signal at N-terminal and several post-translational modifications could be predicted at different residues of the protein. The secondary structure of capsid protein contains coil, beta strand, and alpha helix. The 3D structure of the protein was predicted with a high level of confidence. The capsid protein of PCV4 also contains several potential B-cell epitopes. Thus, the in-silico characterization of the capsid protein will be beneficial in designing diagnostics and vaccine candidates in future to combat PCV4 infection in pigs. [ABSTRACT FROM AUTHOR]

This study investigates the effect of hydraulic shock waves on inactivation of MS-2 bacteriophage, a norovirus surrogate. A falling circular jet of water spiked with the MS-2 (∼1000 PFU/mL) was repeatedly impacted by a rotating blade, resulting in occurrence of hydraulic shock waves within the liquid region adjacent to the impact. The proof-of-concept rotational generator of hydraulic shock treating 9 L of water spiked with viruses was able to achieve 3 logs reduction of viral plaque count within 80–100 liquid passes at moderate blade impact velocities (namely, 70 and 88 m/s) despite the water temperature not exceeding 40 °C and no detectible cavitation. Within the first 20 liquid passes, most MS-2 capsid proteins were degraded, with their concentration reduced from 22 μg/mL to only 7.3 μg/mL. Due to the lack of further capsid protein destruction, additional reduction in MS-2 plaque count in subsequent 80 passes is indicative of damage inflicted to the viral recognition receptors. All this suggests that shockwaves of moderate amplitude (few tens of MPa) alone are sufficient for effective viral inactivation. Considering this and the device's good scalability potential, rotational hydraulic shock generators could prove effective in treating virus-contaminated waters.

Porcine circovirus type 2 (PCV2) has been recognized as a critical pathogen associated with numerous porcine circovirus-related diseases. Immunization is commonly regarded as the most efficient method to combat PCV2 infection. The virus's main antigen is the Cap protein, which is encoded by the ORF2 gene. In this investigation, we cloned the PCV2 Cap gene into the pYA3681 plasmid and subsequently electrotransferred it into the delayed lysis Salmonella strain χ11802, ultimately generating the χ11802 (pYA3681-Cap) vaccine candidate strain. We assessed the levels of sIgA and IgG specific to the Cap protein in mice, revealing that their mucosal and humoral immunity had been activated by χ11802 (pYA3681-Cap). The result was a substantial elevation in antibody levels, and a notable reduction in viral load in the immunized group compared to the unvaccinated group. Furthermore, the study revealed a significant decrease in the viral load in the lungs, liver, and spleen of mice inoculated with χ11802 (pYA3681-Cap), in comparison to both the empty carrier group and the phosphate buffered saline control group. This study further investigated the lytic effect of the delayed lysis vaccine vector. The χ11802 (pYA3681-EGFP) strain was undetectable 10 days post-challenge, indicating that the vaccine strain can effectively release the carried exogenous antigen and prevent the residual vaccine strain from spreading and causing pollution in the environment. A successful construction of the χ11802 (pYA3681-Cap) strain expressing the PCV2 Cap protein was executed in this study. To summarize, our study suggests that PCV2-Cap, when expressed in the delayed lysis Salmonella strain χ11802, could serve as a safe and economically efficient candidate PCV2 vaccine.

Canine circovirus (CanineCV) is a contagious virus that causes severe gastroenteritis, diarrhea, respiratory disease, and vasculitis, often resulting in fatality among infected dogs. In this study, a recombinant Capsid protein (rCap) of CanineCV was expressed in the Escherichia coli (E. coli) Rosetta (DE3) pLysS host cell, followed by affinity purification, and then analyzed by SDSPAGE, revealing a molecular weight of approximately 31 kDa. The antigenicity of the CanineCV rCap protein was confirmed through recognition by a rabbit anti-CanineCV rCap protein polyclonal antibody (PoAb). Additionally, the reactivity and specificity of this PoAb were assessed using indirect enzymelinked immunosorbent assay (ELISA) and Western blot analysis before applying in an immunohistochemistry (IHC), namely, immunoperoxidase detection. The immunoperoxidase assay using rabbit anti-CanineCV rCap protein PoAb demonstrated that the CanineCV Cap protein was predominantly located in immune cells, especially lymphocytes and macrophages, within the spleen, lung, tracheobronchial lymph nodes, small intestine, and kidney. Similarly, the Cap protein was also found in pneumocytes in the lung and renal tubular epithelial cells in the kidney. These findings reflected the biological activity and cell tropism of the virus. Therefore, the recombinant Cap protein and its PoAb could be used for the development of a valuable diagnostic tool for CanineCV detection. [ABSTRACT FROM AUTHOR]

The high genetic heterogeneity of small ruminant lentiviruses (SRLV) renders the genetic characterization of the circulating strains crucial for the epidemiological investigation and the designation of effective diagnostic tools. In Greece, research data regarding the genetic diversity of the circulating SRLV strains is scarce, hindering the implementation of efficient surveillance and control programs. The objective of the study was to genetically characterize SRLV strains isolated from intensive dairy sheep farms in Greece and evaluate the variability of the immunodominant regions of the capsid protein. For this reason, a total of 12 SRLV-infected animals from four intensive dairy sheep farms with purebred Chios and Lacaune ewes were used for the amplification and sequencing of an 800 bp gag-pol fragment. The phylogenetic analyses revealed a breed-related circulation of strains; Chios ewes were infected with strains belonging exclusively to a separate group of genotype A, whereas strains belonging to subtype B2 were isolated from Lacaune ewes. Immunodominant epitopes of capsid protein were quite conserved among the strains of the same genotype, except for the Major Homology Region which showed some unique mutations with potential effects on viral evolution. The present study contributes to the extension of the current knowledge regarding the genetic diversity of SRLV strains circulating in sheep in Greece. However, broader genetic characterization studies are warranted for the exploration of possible recombinant events and the more comprehensive classification of the circulating strains. [ABSTRACT FROM AUTHOR]

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Abstract Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120–134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131–143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120–134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties.

Porcine circovirus type 2 (PCV2) is the etiological agent of PCV2-associated diseases that pose a serious threat to the swine industry. PCV2 capsid (Cap) protein has been shown to interact with DEAD-box RNA helicase 21 (DDX21), an important protein that regulates RNA virus replication. However, whether the interaction between DDX21 and the PCV2 Cap regulates PCV2 replication remains unclear. Herein, by using western blotting, interaction assays, and knockdown analysis, we found that PCV2 infection induced the cytoplasmic relocation of DDX21 from the nucleolus in cultured PK-15 cells. Moreover, the nuclear localization signal (NLS) of PCV2 Cap interacted directly with DDX21. The NLS of PCV2 Cap and 763GSRSNRFQNK772 residues at the C-terminal domain (CTD) of DDX21 were essential for the dual interaction. Upon shRNA-mediated DDX21 depletion in PK-15 cells, we observed impaired PCV2 replication via a lentivirus-delivered system, as evidenced by decreased levels of viral protein expression and virus production. In contrast, the replication of PCV2 increased in transiently DDX21-overexpressing cells. Our results indicate that DDX21 interacts with PCV2 Cap and plays a crucial role in virus replication. These results provide a reference for developing novel potential targets for prevention and control of PCV2 infection. [ABSTRACT FROM AUTHOR]

The Flavivirus genus of the Flaviviridae family of enveloped single-stranded RNA viruses encompasses more than 70 members, many of which cause significant disease in humans and livestock. Packaging and assembly of the flavivirus RNA genome is essential for the formation of virions, which requires intricate coordination of genomic RNA, viral structural, and nonstructural proteins in association with virus-induced, modified endoplasmic reticulum (ER) membrane structures. The capsid (C) protein, a small but versatile RNA-binding protein, and the positive single-stranded RNA genome are at the heart of the elusive flavivirus assembly process. The nucleocapsid core, consisting of the genomic RNA encapsidated by C proteins, buds through the ER membrane, which contains viral glycoproteins prM and E organized as trimeric spikes into the lumen, forming an immature virus. During the maturation process, which involves the low pH-mediated structural rearrangement of prM and E and furin cleavage of prM in the secretory pathway, the spiky immature virus with a partially ordered nucleocapsid core becomes a smooth, mature virus with no discernible nucleocapsid. This review focuses on the mechanisms of genome packaging and assembly by examining the structural and functional aspects of C protein and viral RNA. We review the current lexicon of critical C protein features and evaluate interactions between C and genomic RNA in the context of assembly and throughout the life cycle. [ABSTRACT FROM AUTHOR]

We engineered Saccharomyces cerevisiae to express structural proteins of foot-and-mouth disease virus (FMDV) and produce virus-like particles (VLPs). The gene, which encodes four structural capsid proteins (VP0 (VP4 and VP2), VP3, and VP1), followed by a translational "ribosomal skipping" sequence consisting of 2A and protease 3C, was codon-optimized and chemically synthesized. The cloned gene was used to transform S. cerevisiae 2805 strain. Western blot analysis revealed that the polyprotein consisting of VP0, VP3, and VP1 was processed into the discrete capsid proteins. Western blot analysis of 3C confirmed the presence of discrete 3C protein, suggesting that the 2A sequence functioned as a "ribosomal skipping" signal in the yeast for an internal re-initiation of 3C translation from a monocistronic transcript, thereby indicating polyprotein processing by the discrete 3C protease. Moreover, a band corresponding to only VP2, which was known to be non-enzymatically processed from VP0 to both VP4 and VP2 during viral assembly, further validated the assembly of processed capsid proteins into VLPs. Electron microscopy showed the presence of the characteristic icosahedral VLPs. Our results clearly demonstrate that S. cerevisiae processes the viral structural polyprotein using a viral 3C protease and the resulting viral capsid subunits are assembled into virion particles. Key points: • Ribosomal skipping by self-cleaving FMDV peptide in S. cerevisiae. • Proteolytic processing of a structural polyprotein from a monocistronic transcript. • Assembly of the processed viral capsid proteins into a virus-like particle. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) vector has become the leading platform for gene delivery. Each serotype exhibits a different tissue tropism, immunogenicity, and in vivo transduction performance. Therefore, selecting the most suitable AAV serotype is critical for efficient gene delivery to target cells or tissues. Genome divergence among different serotypes is due mainly to the hypervariable regions of the AAV capsid proteins. However, the heterogeneity of capsid glycosylation is largely unexplored. In the present study, the N-glycosylation profiles of capsid proteins of AAV serotypes 1 to 9 have been systemically characterized and compared using a previously developed high-throughput and high-sensitivity N-glycan profiling platform. The results showed that all 9 investigated AAV serotypes were glycosylated, with comparable profiles. The most conspicuous feature was the high abundance mannosylated N-glycans, including FM3, M5, M6, M7, M8, and M9, that dominated the chromatograms within a range of 74 to 83%. Another feature was the relatively lower abundance of fucosylated and sialylated N-glycan structures, in the range of 23%–40% and 10%–17%, respectively. However, the exact N-glycan composition differed. These differences may be utilized to identify potential structural relationships between the 9 AAV serotypes. The current research lays the foundation for gaining better understanding of the importance of N-glycans on the AAV capsid surface that may play a significant role in tissue tropism, interaction with cell surface receptors, cellular uptake, and intracellular processing. [ABSTRACT FROM AUTHOR]

Successful assembly of bacteriophage MS2 virus-like particles (VLPs) from a secreted recombinant capsid protein in the culture medium of the Komagataella phaffii methylotrophic yeast has been shown. The yield of VLPs was 5 g/L and reached 30% of the total protein of the culture liquid supernatant. The VLPs were well-ordered icosahedral structures with a diameter of 26‒28 nm, which was confirmed by transmission electron microscopy. A simple method for the isolation and purification of the VLPs was proposed, which ensures the purity of the target protein structures of more than 90%. The results we obtained can be used for development of a technology for the large-scale production of recombinant vaccines based on bacteriophage MS2 VLPs. [ABSTRACT FROM AUTHOR]

Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120–134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131–143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120–134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties. [ABSTRACT FROM AUTHOR]

The emerging worldwide distributed porcine circovirus type 3 (PCV3) infection poses a serious threat to swine herds. An important means of preventing and controlling PCV3 infection is the development of the vaccine, while, the inability to cultivate in vitro has become the biggest obstacle. Orf virus (ORFV), the prototypic member of the Parapoxviridae, has been proven to be a novel valid vaccine vector for preparing various candidate vaccines. Here, recombinant ORFV expressing capsid protein (Cap) of PCV3 was obtained and proved its favorable immunogenicity inducing antibody against Cap in BALB/c mice. Based on the enhanced green fluorescent protein (EGFP) as a selectable marker, the recombinant rORFVΔ132-PCV3Cap-EGFP was generated. Then, recombinant ORFV expressing Cap only, rORFVΔ132-PCV3Cap, was obtained based on rORFVΔ132-PCV3Cap-EGFP using a double homologous recombination method by screening single non-fluorescent virus plaque. Results of the western blot showed that the Cap can be detected in rORFVΔ132-PCV3Cap infected OFTu cells. The results of immune experiments in BALB/c mice indicated that a specific antibody against Cap of PCV3 in serum was induced by rORFVΔ132-PCV3Cap infection. The results presented here provide a candidate vaccine against PCV3 and a feasible technical platform for vaccine development based on ORFV. [ABSTRACT FROM AUTHOR]

Norovirus, commonly found on shellfish and vegetables, is a foodborne virus with GII.4 as the dominant genotype responsible for widespread outbreaks since 1995. Continuous variation of major capsid protein VP1 can lead to changes in the immunogenicity and host receptor binding ability of norovirus, which is an important evolutionary mechanism. Therefore, analyzing the immunogenicity of VP1 and its binding ability to various HBGAs in GII.4 variants could improve our understanding of the persistent prevalence of GII.4. Here, the results suggest that GII.4 has gradually enhanced its HBGAs binding ability over time for various types of receptors. Variants exhibit significantly stronger immune response to homologous mouse antiserum than heterologous ones, highlighting the importance of variation of antigenic and histo-blood group binding sites in driving the evolution of GII.4. These synergistic forces constantly lead to antigenic drift and changes in receptor binding, resulting in continuous emergence of new variant strains and sustained prevalence.

Despite the global implementation of prequalified oral vaccines by the World Health Organization (WHO) in numerous countries, rotavirus A (RVA) have continued to be the principal cause of acute dehydrating diarrhoea in children under five years of age. Unfortunately, there are no authorized anti-rotaviral medications. Hence, it is crucial to prioritize the development of specialized therapeutics to combat rotaviral infections. For the first time, Spondias mombin, Macaranga barteri and Dicerocaryum eriocarpum metabolites were screened using computational techniques to identify potential novel modulators with broad-spectrum activity against VP7 epitopes (capsid protein) of RVA. Compounds with poor pharmacokinetics and drug-likeness were screened out from the initial top 20 metabolites identified using molecular docking. Thereafter, molecular dynamics (MD) simulation was used to assess the ability of the resulting compounds to modulate the selected VP7 epitopes. Remarkably, all the lead compounds had higher negative binding free energy than tizoxanide across the three epitopes of VP7, with apigenin-4′-glucoside having the highest affinity for VP7A (−24.13 kcal/mol) and VP7C (−43.67 kcal/mol) while the highest affinity for VP7D was observed in 2SG (−36.08 kcal/mol). Interestingly, 2SG (−18.24 kcal/mol, −31.21 kcal/mol, −36.08 kcal/mol), apigenin-4′-glucoside (−24.13 kcal/mol, −43.67 kcal/mol, −33.52 kcal/mol) and gnetin L (−21.21 kcal/mol, −27.56 kcal/mol, −32.48 kcal/mol) had better broad-spectrum affinities for VP7A, C and D relative to tizoxanide (−10.36 kcal/mol, −18.32 kcal/mol, −12.98 kcal/mol) respectively. Generally, the compounds are thermodynamically stable with 2SG (1.88 Å), ellagic acid (2.45 Å) and sericetin (2.22 Å) being the most stable against VP7A, C and D respectively. While this study unearths the lead compounds' promising ability to modulate the investigated VP7 epitopes, further confirmatory in vitro and in vivo studies are imperative, and efforts are underway towards achieving this task.

The zoonotic transmission of hepatitis E virus (HEV) genotypes 3 (HEV-3) and 4 (HEV-4), and rabbit HEV (HEV-3ra) has been documented. Vaccination against HEV infection depends on the capsid (open reading frame 2, ORF2) protein, which is highly immunogenic and elicits effective virus-neutralizing antibodies. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles (VLPs). However, research on the production of ORF2 proteins from these HEV genotypes in E. coli to form VLPs has been modest. In this study, we constructed 21 recombinant plasmids expressing various N-terminally and C-terminally truncated HEV ORF2 proteins for HEV-3, HEV-3ra, and HEV-4 in E. coli. We successfully obtained nine HEV-3, two HEV-3ra, and ten HEV-4 ORF2 proteins, which were primarily localized in inclusion bodies. These proteins were solubilized in 4 M urea, filtered, and subjected to gel filtration. Results revealed that six HEV-3, one HEV-3ra, and two HEV-4 truncated proteins could assemble into VLPs. The purified VLPs displayed molecular weights ranging from 27.1 to 63.4 kDa and demonstrated high purity (74.7–95.3%), as assessed by bioanalyzer, with yields of 13.9–89.6 mg per 100 mL of TB medium. Immunoelectron microscopy confirmed the origin of these VLPs from HEV ORF2. Antigenicity testing indicated that these VLPs possess characteristic HEV antigenicity. Evaluation of immunogenicity in Balb/cAJcl mice revealed robust anti-HEV IgG responses, highlighting the potential of these VLPs as immunogens. These findings suggest that the generated HEV VLPs of different genotypes could serve as valuable tools for HEV research and vaccine development.

The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein.

Brevipalpus-transmitted viruses (BTVs) have a significant negative economic impact on the citrus industry in Central and South America. Until now, only a few studies have explored the intracellular distribution and interaction of BTVs-encoded proteins with host factors, particularly for cileviruses, the main BTV responsible for the Citrus Leprosis (CL) disease. This study describes the nuclear localization of citrus leprosis virus C (CiLV-C) capsid protein (p29) and its interaction with the fibrillarin (Fib2) within the nucleolar compartment and cell cytoplasm. Our results, obtained by computer predictions and laser scanning confocal microscopy analyses, including colocalization and bimolecular fluorescence complementation (BiFC) approaches, revealed that a fraction of the p29 is localized in the nucleus and colocalizes with the Fib2 in both the nucleolus and cytosol. The nuclear localization of p29 correlated with a smaller nucleus size. Furthermore, co-immunoprecipitation (Co-IP) assays confirmed the interactions between p29 and Fib2. The implications of these findings for the functionalities of the cilevirus capsid protein are discussed.

Canine parvovirus (CPV-2) is one of the most important pathogens of dogs of all ages, causing pandemic infections that are characterized by fatal hemorrhagic enteritis. The CPV-2 vaccine is recommended as a core vaccine for pet animals. Despite the intensive practice of active immunization, CPV-2 remains a global threat. In this study, a multi-epitope vaccine against CPV-2 was designed, targeting the highly conserved capsid protein (VP2) via in silico approaches. Several immunoinformatics methods, such as epitope screening, molecular docking, and simulation were used to design a potential vaccine construct. The partial protein sequences of the VP2 gene of CPV-2 and protein sequences retrieved from the NCBI were screened to predict highly antigenic proteins through antigenicity, trans-membrane-topology screening, an allergenicity assessment, and a toxicity analysis. Homologous VP2 protein sequences typically linked to the disease were identified using NCBI BLAST, in which four conserved regions were preferred. Overall, 10 epitopes, DPIGGKTGI, KEFDTDLKP, GTDPDDVQ, GGTNFGYIG, GTFYFDCKP, NRALGLPP, SGTPTN, LGLPPFLNSL, IGGKTG, and VPPVYPN, were selected from the conserved regions to design the vaccine construct. The molecular docking demonstrated the higher binding affinity of these epitopes with dog leukocyte antigen (DLA) molecules. The selected epitopes were linked with Salmonella enterica flagellin FliC adjuvants, along with the PADRE sequence, by GGS linkers to construct a vaccine candidate with 272 nucleotides. The codon adaptation and in silico cloning showed that the generated vaccine can be expressed by the E. coli strain, K12, and the sequence of the vaccine construct showed no similarities with dog protein. Our results suggest that the vaccine construct might be useful in preventing canine parvoviral enteritis (CPE) in dogs. Further in vitro and in vivo experiments are needed for the validation of the vaccine candidate. [ABSTRACT FROM AUTHOR]

Introduction: Duck circovirus (DuCV) infection is currently recognized as an important immunosuppressive disease in commercial duck flocks in China. Specific antibodies against DuCV viral proteins are required to improve diagnostic assays and understand the pathogenesis of DuCV infection. Methodsand results: To generate DuCV-specific monoclonal antibodies (mAbs), a recombinant DuCV capsid protein without the first 36 N-terminal amino acids was produced in Escherichia coli. Using the recombinant protein as an immunogen, a mAb was developed that reacted specifically with the DuCV capsid protein, expressed in E. coli and baculovirus systems. Using homology modeling and recombinant truncated capsid proteins, the antibody-binding epitope was mapped within the region of 144IDKDGQIV151, which is exposed to solvent in the virion capsid model structure. To assess the applicability of the mAb to probe the native virus antigen, the murine macrophage cell line RAW267.4 was tested for DuCV replicative permissiveness. Immunofluorescence and Western blot analysis revealed that the mAb recognized the virus in infected cells and the viral antigen in tissue samples collected from clinically infected ducks. Discussion: This mAb, combined with the in vitro culturing method, would have widespread applications in diagnosing and investigating DuCV pathogenesis. [ABSTRACT FROM AUTHOR]

Genome packaging is the crucial step for maturation of plant viruses containing an RNA genome. Viruses exhibit a remarkable degree of packaging specificity, despite the probability of co-packaging cellular RNAs. Three different types of viral genome packaging systems are reported so far. The recently upgraded type I genome packaging system involves nucleation and encapsidation of RNA genomes in an energy-dependent manner, which have been observed in most of the plant RNA viruses with a smaller genome size, while type II and III packaging systems, majorly discovered in bacteriophages and large eukaryotic DNA viruses, involve genome translocation and packaging inside the prohead in an energy-dependent manner, i.e., utilizing ATP. Although ATP is essential for all three packaging systems, each machinery system employs a unique mode of ATP hydrolysis and genome packaging mechanism. Plant RNA viruses are serious threats to agricultural and horticultural crops and account for huge economic losses. Developing control strategies against plant RNA viruses requires a deep understanding of their genome assembly and packaging mechanism. On the basis of our previous studies and meticulously planned experiments, we have revealed their molecular mechanisms and proposed a hypothetical model for the type I packaging system with an emphasis on smaller plant RNA viruses. Here, in this review, we apprise researchers the technical breakthroughs that have facilitated the dissection of genome packaging and virion assembly processes in plant RNA [ABSTRACT FROM AUTHOR]

Dengue virus belongs to the Flaviviridae family, being responsible for an endemic arboviral disease in humans. It is an enveloped virus, whose genome is a positive-stranded RNA packaged by the capsid protein. Dengue virus capsid protein (DENVC) forms homodimers in solution organized in 4 α-helices and an intrinsically disordered N-terminal region. The N-terminal region is involved in the binding of membranous structures in host cells and in the recognition of nucleotides. Here we report the 1H, 15N and 13C resonance assignments of the DENVC with the deletion of the first 19 intrinsically disordered residues. The backbone chemical shift perturbations suggest changes in the α1 and α2 helices between full length and the truncated proteins. [ABSTRACT FROM AUTHOR]

Objective To prepare polyclonal antisera against enterovirus 71(EV71) capsid proteins. Methods Five B cell epitope prediction softwares were used to analyze capsid proteins of EV71 and four peptides against viral protein were synthesized, VP1(aa.204-223), VP2(aa.133-163), VP3(aa.139-148+aa.173-191) and VP4(aa.1-23). They were conjugated with keyhole limpet hemocyanin(KLH). BALB/c mice were subcutaneously immunized with six doses of KLH-peptides adjuvanted with MF59/CpGC274 at two-week interval. Serum sample was collected and examined with ELISA and Western blot. Results The four antisera were bound to both EV71 virus and denatured virus effectively. VP2 antiserum showed activity with denatured virus and the other three had similar binding activity to EV71 virus and denatured virus. The four antiserum samples were bound to both EV71 virus-like particle (VLP) and denatured VLP effectively. VP1 and VP2 antiserum could bind to VP1, VP0 of EV71 VLP separately. VP2 antiserum showed the highese binding activity and VP1 antiserum showed a inperior binding activity as compared to the formers. VP1 and VP2 antiserum were bound specifically and efficiently to EV71 virus. VP0 of empty particles and VP2 of intact EV71 virus particles could be simultaneously identified by VP2 antiserum. VP3 and VP4 antiserum failed to binding to no obvious binding to VLP and virus. Conclusions Four epitope peptide polyclonal antisera against EV71 are successfully prepared, which can provide ootential detection tools used in ELISA and Western blot and may be used for QC evaluation of EV71 vaccine.

Abstract Background The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host’s immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. Results The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. Conclusions This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.

Macroautophagy/autophagy is a conserved mechanism launched by host organisms to fight against virus infection. Double-membraned autophagosomes in arthropod vectors can be remodeled by arboviruses to accommodate virions and facilitate persistent viral propagation, but the underlying mechanism is unknown. Rice gall dwarf virus (RGDV), a plant nonenveloped double-stranded RNA virus, induces the formation of virus-containing double-membraned autophagosomes to benefit persistent viral propagation in leafhopper vectors. In this study, it was found that the capsid protein P2 of RGDV alone induced autophagy. P2 specifically interacted with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and ATG4B both in vitro and in vivo. Furthermore, the GAPDH-ATG4B complex could be recruited to virus-induced autophagosomes. Silencing of GAPDH or ATG4B expression suppressed ATG8 lipidation, autophagosome formation, and efficient viral propagation. Thus, P2 could directly recruit the GAPDH-ATG4B complex to induce the formation of initial autophagosomes. Furthermore, such autophagosomes were modified to evade fusion with lysosomes for degradation, and thus could be persistently exploited by viruses to facilitate efficient propagation. GAPDH bound to ATG14 and inhibited the interaction of ATG14 with SNAP29, thereby preventing ATG14-SNARE proteins from mediating autophagosome-lysosome fusion. Taken together, these results highlight how RGDV activates GAPDH to initiate autophagosome formation and block autophagosome degradation, finally facilitating persistent viral propagation in insect vectors. The findings reveal a positive regulation of immune response in insect vectors during viral infection. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is the notable causative agent of acute and chronic hepatic, renal, pancreatic, neurological, and hematopoietic blood cell infections with high risk in immunocompromised patients. Hepatic failure is mostly documented among adults, pregnant women, and patients with preexisting liver disease. HEV is a positive sense RNA virus of 7.2 kb genome size with typically three open reading frames (ORFs) which play essential roles in viral replication, genome assembly, and transcription. The mutational substitution in the viral RNA genome makes more it difficult to understand the actual relationship in the host–virus association. ORFs of HEV encode different structural and non-structural proteins and one of them is the capsid protein which is coded by ORF2. The capsid protein mediates the encapsulation of the viral genome as well as being involved in virion assembly. In the current study, the ligand-based docking approach was employed to inhibit the active amino acids of the viral capsid protein. Depending upon S-score, ADMET profiling, and drug scanning, the top ten tetrapeptides were selected as potential drug candidates with no toxicity counter to HEV receptor protein. The S-score or docking score is a mathematical function which predicts the binding affinities of docked complexes. The binding affinity of the predicted drug–target complexes helps in the selectivity of the desired compound as a potential drug. The best two selected peptides (i.e., TDGH with S-score of −8.5 and EGDE with S-score of −8.0) interacted with the active site amino acids of the capsid protein (i.e., Arg399, Gln420, and Asp444). The molecular dynamics simulations of RMSD trajectories of TDGH–capsid protein and EDGE–capsid protein have revealed that both docked complexes were structurally stable. The study revealed that these tetrapeptides would serve as strong potential inhibitors and a starting point for the development of new drug molecules against the HEV capsid protein. In future, in vivo studies are needed to explore selected peptides as potential drug candidates. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses (SRLV) infect sheep and goats resulting in significant economic losses. This study evaluated for the first time the predicted conformational structure of the SRLV-capsid-protein 25 (SRLV-p25) and analyzed the antigenicity of recombinant protein (SRLV-rp25) in mice by coupling to an immunostimulatory complexes based on glycyrrhizinic acid liposomes (GAL) and tested plasma from goats and sheep naturally infected. Analysis in silico and conformational structure of SRLV-p25 (genotype B-FESC-752) showed similar characteristics to other lentiviral capsids. The efficient expression of SRLV-rp25 was confirmed by Western blot. The humoral immune responses in mice showed an increased level of antibodies from day 21 to 35 of the SRLV-r p25-GAL and SRLV-rp25-ISCOM® groups and the cellular immune response showed no significant difference in IL-10 levels (P > .05), however, a significant difference (P < .001) was observed when comparing SRLV-rp25-GAL with SRLV-rp25 groups. Immunoreactivity toward SRLV- r p25 revealed 61% of positive samples from naturally infected goats and sheep. [ABSTRACT FROM AUTHOR]

Background: Feline panleukopenia virus (FPV) is a widespread and highly infectious pathogen in cats with a high mortality rate. Although Yanji has a developed cat breeding industry, the variation of FPV locally is still unclear. Objectives: This study aimed to isolate and investigate the epidemiology of FPV in Yanji between 2021 and 2022. Methods: A strain of FPV was isolated from F81 cells. Cats suspected of FPV infection (n = 80) between 2021 and 2022 from Yanji were enrolled in this study. The capsid protein 2 (VP2) of FPV was amplified. It was cloned into the pMD-19T vector and transformed into a competent Escherichia coli strain. The positive colonies were analyzed via VP2 Sanger sequencing. A phylogenetic analysis based on a VP2 coding sequence was performed to identify the genetic relationships between the strains. Results: An FPV strain named YBYJ-1 was successfully isolated. The virus diameter was approximately 20-24 nm, 50% tissue culture infectious dose = 1 × 10-4.94/mL, which caused cytopathic effect in F81 cells. The epidemiological survey from 2021 to 2022 showed that 27 of the 80 samples were FPV-positive. Additionally, three strains positive for CPV-2c were unexpectedly found. Phylogenetic analysis showed that most of the 27 FPV strains belonged to the same group, and no mutations were found in the critical amino acids. Conclusions: A local FPV strain named YBYJ-1 was successfully isolated. There was no critical mutation in FPV in Yanji, but some cases with CPV-2c infected cats were identified. [ABSTRACT FROM AUTHOR]

Goose astrovirus (GAstV) was classified into GAstV-1 and GAstV-2, and both caused gosling viral gout. Recently, there has been no effective commercial vaccine to control the infection. It is important to establish serological methods to distinguish between the two genotypes. In this study, we reported the development and application of two indirect enzyme-linked immunosorbent assays (ELISAs) using the GAstV-1 virus and a recombinant GAstV-2 capsid protein as specific antigens to detect antibodies against GAstV-1 and GAstV-2, respectively. The optimal coating antigen concentration of indirect GAstV-1-ELISA and GAstV-2-Cap-ELISA was 1.2 µg/well and 125 ng/well, respectively. In addition, the antigen coating temperature and time, sera dilution and reaction time, and the dilution and reaction time of HRP-conjugated secondary antibody were optimized. The cut-off values were 0.315 and 0.305, and the analytical sensitivity was 1:6400 and 1:3200 for indirect GAstV-1-ELISA and GAstV-2-Cap-ELISA, respectively. The assays were able to differentiate specific sera against GAstVs, TUMV, GPV, and H9N2-AIV. The intra- and inter-plate variabilities of indirect ELISAs were less than 10%. The coincidence rate of positive sera was higher than 90%. The indirect ELISAs were further applied to test 595 goose serum samples. The results showed that the detection rates were 33.3% and 71.4% in GAstV-1-ELISA and GAstV-2-Cap-ELISA, respectively, and the co-detection rate was 31.1%, which indicates that the seroprevalence rate of GAstv-2 was higher than that of GastV-1, and the co-infection existed between GAstV-1 and GAstV-2. In summary, the developed GAstV-1-ELISA and GAstV-2-Cap-ELISA have high specificity, sensitivity, and reproducibility and can be used in the clinical detection of the antibody against GAstV-1 and GAstV-2. [ABSTRACT FROM AUTHOR]

ABSTRACT Porcine circovirus type 2 (PCV2) is an important swine infectious pathogen that seriously threatens the global swine industry. PCV2 Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. In this study, a gold nanoparticle-based immunochromatographic strip with high sensitivity and specificity was developed which could be used for rapid detection of PCV2 virions or Cap protein in research. The visual detection limit of the strip was 103.18 50% tissue culture infective does (TCID50)/mL for PCV2, and 2.03 μg/mL for PCV2 Cap protein. No cross-reactivity was observed with the PCV1 and PCV3 Cap proteins and other common swine pathogens such as porcine reproductive and respiratory syndrome virus, classical swine fever virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine parvovirus, and swine influenza virus. The repeatability of the strip was good. The stability of the strip was perfect for 12 months in a dry state at room temperature. Visual results could be obtained within 5 min by simply inserting the strip into the diluted sample. The strip is a time-saving, labor-saving, and reliable tool for testing of PCV2 virions or Cap protein in research. The idea of this study might open a new perspective for the application of the strip. IMPORTANCE Porcine circovirus type 2 (PCV2) Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. Although many methods can be used to identify PCV2 or PCV2 Cap protein in vaccine research, they usually require high workload and time. The developed strip can specifically detect PCV2 virions or Cap protein, and visual qualitative results can be obtained within 5 min by simply diluting the sample and inserting the strip into the sample. The final value of the strip is providing a simple and time-saving method for real-time monitoring of PCV2 antigen in vaccine research with reliable results, such as the different stages of PCV2 Cap protein expression and purification, as well as the different stages of PCV2 reproduction and purification.

Genome packaging is the crucial step for maturation of plant viruses containing an RNA genome. Viruses exhibit a remarkable degree of packaging specificity, despite the probability of co-packaging cellular RNAs. Three different types of viral genome packaging systems are reported so far. The recently upgraded type I genome packaging system involves nucleation and encapsidation of RNA genomes in an energy-dependent manner, which have been observed in most of the plant RNA viruses with a smaller genome size, while type II and III packaging systems, majorly discovered in bacteriophages and large eukaryotic DNA viruses, involve genome translocation and packaging inside the prohead in an energy-dependent manner, i.e., utilizing ATP. Although ATP is essential for all three packaging systems, each machinery system employs a unique mode of ATP hydrolysis and genome packaging mechanism. Plant RNA viruses are serious threats to agricultural and horticultural crops and account for huge economic losses. Developing control strategies against plant RNA viruses requires a deep understanding of their genome assembly and packaging mechanism. On the basis of our previous studies and meticulously planned experiments, we have revealed their molecular mechanisms and proposed a hypothetical model for the type I packaging system with an emphasis on smaller plant RNA viruses. Here, in this review, we apprise researchers the technical breakthroughs that have facilitated the dissection of genome packaging and virion assembly processes in plant RNA viruses.

The Flavivirus genus of the Flaviviridae family of enveloped single-stranded RNA viruses encompasses more than 70 members, many of which cause significant disease in humans and livestock. Packaging and assembly of the flavivirus RNA genome is essential for the formation of virions, which requires intricate coordination of genomic RNA, viral structural, and nonstructural proteins in association with virus-induced, modified endoplasmic reticulum (ER) membrane structures. The capsid (C) protein, a small but versatile RNA-binding protein, and the positive single-stranded RNA genome are at the heart of the elusive flavivirus assembly process. The nucleocapsid core, consisting of the genomic RNA encapsidated by C proteins, buds through the ER membrane, which contains viral glycoproteins prM and E organized as trimeric spikes into the lumen, forming an immature virus. During the maturation process, which involves the low pH-mediated structural rearrangement of prM and E and furin cleavage of prM in the secretory pathway, the spiky immature virus with a partially ordered nucleocapsid core becomes a smooth, mature virus with no discernible nucleocapsid. This review focuses on the mechanisms of genome packaging and assembly by examining the structural and functional aspects of C protein and viral RNA. We review the current lexicon of critical C protein features and evaluate interactions between C and genomic RNA in the context of assembly and throughout the life cycle.

Genome packaging is a critical step during the viral maturation process. Viruses employ a distinct approach to package their genetic materials inside capsid: ranging from very simple strategy of nucleation of capsid proteins onto genome to complex segro-packasome machinery. The majority of small plant viruses, which falls under type I passive system, package their genome into stable virions in the cytoplasmic compartment, where chances of co-packaging of host RNA is very high, indicates viruses evolved the mechanism of selective and stringent packaging of their genomes. Recent discoveries of the unique ATPase fold in the capsid proteins of smaller plant viruses and their direct or indirect role during genome packaging have changed the perception about genome packaging in type I system. We feel that viruses of type I system have acquired unique and independent innovations for genome packaging over the course of evolution. The molecular interactions, intriguingly, cross-talk between capsid proteins and conserved signal sequence situated at the end of genome, plays an important role while viral genome packaging and translocation. Strategy utilized by smaller plant viruses for enhancement of selective genome packaging does not depend simply on nucleation of capsid proteins over genome but interestingly, configuration of viral genome, replicase, tRNA, viral encoded movement proteins are the other important key players that regulate genome packaging. The main aim of this review is to discuss and revisit the mechanism of genome packaging among viruses of agronomic importance. This study will be also useful for understanding the origin and evolution of viral genome packaging apparatuses and their roles in eukaryogenesis. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is a highly infectious and lethal double-stranded DNA virus that is responsible for African swine fever (ASF). ASFV was first reported in Kenya in 1921. Subsequently, ASFV has spread to countries in Western Europe, Latin America, and Eastern Europe, as well as to China in 2018. ASFV epidemics have caused serious pig industry losses around the world. Since the 1960s, much effort has been devoted to the development of an effective ASF vaccine, including the production of inactivated vaccines, attenuated live vaccines, and subunit vaccines. Progress has been made, but unfortunately, no ASF vaccine has prevented epidemic spread of the virus in pig farms. The complex ASFV structure, comprising a variety of structural and non-structural proteins, has made the development of ASF vaccines difficult. Therefore, it is necessary to fully explore the structure and function of ASFV proteins in order to develop an effective ASF vaccine. In this review, we summarize what is known about the structure and function of ASFV proteins, including the most recently published findings. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito‐borne RNA virus that belongs to the Flaviviridae family. While flavivirus replication is known to occur in the cytoplasm, a significant portion of the viral capsid protein localizes to the nucleus during infection. However, the role of the nuclear capsid is less clear. Herein, we demonstrated SERTA domain containing 3 (SERTAD3) as an antiviral interferon stimulatory gene product had an antiviral ability to ZIKV but not JEV. Mechanistically, we found that SERTAD3 interacted with the capsid protein of ZIKV in the nucleolus and reduced capsid protein abundance through proteasomal degradation. Furthermore, an eight amino acid peptide of SERTAD3 was identified as the minimum motif that binds with ZIKV capsid protein. Remarkably, the eight amino acids synthetic peptide from SERTAD3 significantly prevented ZIKV infection in culture and pregnant mouse models. Taken together, these findings not only reveal the function of SERTAD3 in promoting proteasomal degradation of a specific viral protein but also provide a promising host‐targeted therapeutic strategy against ZIKV infection. [ABSTRACT FROM AUTHOR]

BACKGROUND & OBJECTIVE: Dengue virus (DENV) genomic study is crucial for understanding how it spreads and presents and becomes dangerous. Three structural and seven non-structural proteins are encoded by the positive-stranded RNA DENV. Capsid protein (C) is one of the structural proteins which helps in the encapsidation of viral RNA. The objective of the study is to identify the DENV serotypes and the most common geographic specific mutations in the C protein circulating in the Punjab province of Pakistan. METHODOLOGY: It was a cross-sectional study in which about 120 DENV isolates were selected by the procedure of temporal sampling done in the peak season of dengue fever in 2022 from the major tertiary care hospitals for whole genomes sequence analysis. Among these only 23 whole genome sequences were performed after virus isolation, quantification, and cDNA synthesis. RESULTS: All the samples were found to be of genotypes 1 and 2, in which 44 non-synonymous mutations were detected in the C protein. The most common mutations were; N90S (n=11) and G70S (n=11) followed by V26G (n=5) and R10Q (n=4), in which G70S is novel. CONCLUSION: Mutations in the current study are of particular interest to the design of DENV vaccine projects in the future. Genomic epidemiology during each outbreak of DENV in different locations is critical for better public health and for designing new policies for future outbreaks. [ABSTRACT FROM AUTHOR]

T Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122GYVAGFL128 and 227SLYNDSL233. Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122GYVAGFL128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227SLYNDSL233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227SLYNDSL233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122GYVAGFL128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures. [ABSTRACT FROM AUTHOR]

Nuclear entrance and stability of porcine circovirus type 2 (PCV2), the smallest virus in mammals, are crucial for its infection and replication. However, the mechanisms are not fully understood. Here, we found that the PCV2 virion maintains self-stability via the host importin 5 (IPO5) during infection. Coimmunoprecipitation combined with mass spectrometry and glutathione S-transferase pulldown assays showed that the capsid protein (Cap) of PCV2 binds directly to IPO5. Fine identification demonstrated that the N-terminal residue arginine24 of Cap is the most critical to efficient binding to the proline709 residue of IPO5. Detection of replication ability further showed that IPO5 supports PCV2 replication by promoting the nuclear import of incoming PCV2 virions. Knockdown of IPO5 delayed the nuclear transport of incoming PCV2 virions and significantly decreased the intracellular levels of overexpressed PCV2 Cap, which was reversed by treatment with a proteasome inhibitor or by rescuing IPO5 expression. Cycloheximide treatment showed that IPO5 increases the stability of the PCV2 Cap protein. Taken together, our findings demonstrated that during infection, IPO5 facilitates PCV2 replication by directly binding to the nuclear localization signal of Cap to block proteasome degradation. IMPORTANCE Circovirus is the smallest virus to cause immune suppression in pigs. The capsid protein (Cap) is the only viral structural protein that is closely related to viral infection. The nuclear entry and stability of Cap are necessary for PCV2 replication. However, the molecular mechanism maintaining the stability of Cap during nuclear trafficking of PCV2 is unknown. Here, we report that IPO5 aggregates within the nuclear periphery and combines with incoming PCV2 capsids to promote their nuclear entry. Concurrently, IPO5 inhibits the degradation of newly synthesized Cap protein, which facilitates the synthesis of virus proteins and virus replication. These findings highlight a mechanism whereby IPO5 plays a dual role in PCV2 infection, which not only enriches our understanding of the virus replication cycle but also lays the foundation for the subsequent development of antiviral drugs. [ABSTRACT FROM AUTHOR]

Background: Trichomonas vaginalis is the causative agent of a sexually transmitted disease in humans. The virulence of the parasite depends on multiple factors, including the presence of endosymbiotic dsRNA viruses. The presence of Trichomonasviruses (TVV) was associated with more severe genital symptoms, increased proinflammatory host reactions, and modulated parasite sensitivity to metronidazole. However, no efficient antiviral drugs are available against TVV to derive isogenic TVV-positive and TVV-negative cell lines that are essential for investigations of the TVV impact on T. vaginalis biology. Methods: 7-Deaza-2′-C-methyladenosine (7d2CMA) and 2′-C-methylcytidine (2CMC) were used for TVV inhibitory assay. TVV replication was monitored using quantitative reverse transcription PCR (RT qPCR) and western blotting. Modeling of TVV1 RNA-dependent RNA polymerase (RdRp) was performed to visualize the inhibitor-RdRp interaction. Susceptibility to metronidazole was performed under aerobic and anaerobic conditions. Results: We demonstrated that 2CMC but not 7d2CMA is a potent inhibitor of TVV replication. Molecular modeling suggested that the RdRp active site can accommodate 2CMC in the active triphosphate nucleotide form. The effect of 2CMC was shown on strains infected with a single and multiple TVV species. The optimal 2CMC concentration (10 μM) demonstrated strong selectivity for TVVs over trichomonad growth. The presence of TVV has no effect on T. vaginalis metronidazole susceptibility in derived isogenic cell lines. Conclusions: 2CMC acts against TVVs and represents a new inhibitor against Totiviridae viruses. Our isogenic clones are now available for further studies of various aspects of T. vaginalis biology related to TVV infection.

Nervous necrosis virus (NNV) capsid protein plays an important role in producing viral particles without any genetic elements. Thus, NNV is a promising candidate for vaccine development and is widely used for constructing vaccines, including DNA, recombinant proteins, and virus-like particles (VLPs). Our study aimed to investigate the potential of NNV capsid protein (NNV) and NNV capsid protein fused to enhanced green fluorescent protein (NNV-EGFP) through VLP formation and whether their application can induce specific antibody responses against certain antigens. We focused on producing DNA and recombinant protein vaccines consisting of the genes for NNV, EGFP, and NNV-EGFP. The approach using NNV-EGFP allowed NNV to act as a carrier or inducer while EGFP was incorporated as part of the capsid protein, thereby enhancing the immune response. In vitro studies demonstrated that all DNA vaccines expressed in HINAE cells resulted in varying protein expression levels, with particularly low levels observed for pNNV and pNNV-EGFP. Consequently, structural proteins derived from HINAE cells could not be observed using transmission electron microscopy (TEM). In contrast, recombinant proteins of NNV and NNV-EGFP were expressed through the Escherichia coli expression system. TEM revealed that rNNV was assembled into VLPs with an approximate size of 30 nm, whereas rNNV-EGFP presented particles ranging from 10 nm to 50 nm in size. For the vaccination test, DNA vaccination marginally induced specific antibody responses in Japanese flounder compared to unvaccinated fish. Meanwhile, NNV and NNV-EGFP recombinant vaccines enhanced a greater anti-NNV antibody response than the others, whereas antibody responses against EGFP were also marginal. These results indicate that NNV capsid protein-based antigens, presenting as particles, play an important role in eliciting a specific anti-NNV antibody response and have the potential to improve fish immune responses. • Using NNV capsid protein as a carrier to deliver EGFP and enhance immune responses. • The E. coli -derived rNNV and rNNV-EGFP emerged as virus-like structures. • Antigen-presenting particles elicited the anti-NNV antibody response. [ABSTRACT FROM AUTHOR]

BACKGROUND & OBJECTIVE: Dengue virus (DENV) genomic study is crucial for understanding how it spreads and presents and become dangerous. Three structural and seven non-structural proteins are encoded by the positive stranded RNA DENV. Caspid protein (C) is one of the structural proteins which helps in the encapsidation of viral RNA. Objective of the study is to identify the DENV serotypes and the most common geographic specific mutations in the C protein, circulating in Punjab province of Pakistan. METHODOLOGY: It was cross sectional study in which About 120 DENV isolates were selected, by the procedure of temporal sampling done in peak season of dengue fever in 2022, from the major tertiary care hospitals for whole genomes sequence analysis. Among which only 23 whole genomes sequences were performed after virus isolation, quantification and cDNA synthesis. RESULTS: All the samples were found to be of genotype 1 and 2 in which 44 non-synonymous mutations were detected in the C protein. The most common mutations were; N90S (n=11) and G70S (n=11) followed by V26G (n=5) and R10Q (n=4), in which G70S is novel. CONCLUSION: Mutations in the current study are of particular interest to design of DENV vaccine projects in future. Genomic epidemiology during each outbreak of DENV in different locations is critical for better public health and for designing new policies for future outbreaks.

Porcine circovirus type 2 (PCV2) is capable of causing porcine circovirus-associated disease (PCVAD) and is one of the major threats to the global pig industry. The nucleocapsid protein Cap encoded by the PCV2 ORF2 gene is an ideal antigen for the development of PCV2 subunit vaccines, and its N-terminal nuclear localization sequence (NLS) structural domain is essential for the formation of self-assembling VLPs. In the present study, we systematically expressed and characterized full-length PCV2 Cap proteins fused to dominant T and B cell antigenic epitopes and porcine-derived CD154 molecules using baculovirus and found that the Cap proteins fusing epitopes were still capable of forming virus-like particles (VLPs). Both piglet and mice experiments showed that the Cap proteins fusing epitopes or paired with the molecular adjuvant CD154 were able to induce higher levels of humoral and cellular responses, particularly the secretion of PCV2-specific IFN-γ and IL-4. In addition, vaccination significantly reduced clinical signs and the viral load of PCV2 in the blood and tissues of challenged piglets. The results of the study provide new ideas for the development of a more efficient, safe and broad-spectrum next-generation PCV2 subunit vaccine. [ABSTRACT FROM AUTHOR]